The abuse of the amphetamine analog, 3,4-methylenedioxymethamphetamine (MDMA), continues as a significant public health problem. Treatment of rats with MDMA destroys brain serotonergic nerve terminals which is accompanied by a long-lasting decrease in tryptophan hydroxylase (TPH) activity. The mechanism by which this neuronal damage occurs remains unknown. Interestingly, MDMA causes a biphasic response in the central serotonergic system. The first phase consists of a rapid decrease in the cerebral TPH activity which is detected within 15 min of drug administration. This early phase (phase l) is reversed by incubating the enzyme in vitro under reducing conditions. In the second phase, a decrease in TPH activity (phase 2) is observed days after MDMA administration and last for extended periods of time; in contrast to phase l, phase 2 cannot be reversed by reducing conditions. The long-lasting change in TPH activity is associated with MDMA-induced neurotoxicity. Evidence presented in this proposal suggests that TPH protein undergoes an initial reversible transformation during phase 1, with other possible transformations during an intermediate transitional stage occurring prior to TPH protein destruction due to neuronal damage (phase 2). The phase l decrease in TPH activity is caused by oxidative conditions which are proposed to predispose the serotonergic neuron to permanent neuronal (damage. Since TPH is sensitive to MDMA-induced oxidative alterations present in the serotonergic system, we propose to use this enzyme as a probe to monitor and characterize this phenomenon. The characterization of these oxidative conditions will be achieved by identifying the different stages of TPH protein transformation by correlating the MDMA-induced decrease in TPH activity with its protein concentration and with recovery of enzyme activity induced by reducing conditions. The MDMA-generated oxidative conditions will be further characterized by studying the effects of different free radical scavengers on the three stages of TPH transformation. The role of glutathione as a protective system in the serotonergic system will also be investigated. Finally, the reactive substances generated by MDMA will be investigated by studying the alterations of the TPH protein isolated during the different stages by immunoprecipitation and electrophoresis. This study should assist in identifying the biochemical alterations induced by MDMA and similar amphetamine analogs and their impact on brain proteins.